Study On The Mechanism Of Apoptosis Induced By Salidroside In Human HCC HepG2 Cells

Objective:Traditional Chinese medicine plays a key role in the treatment of cancers. Extraction of active components from herbal and natural medicines and evaluation of their efficacy on tumors have been an active research area in current pharmacology. Salidroside, the primary active component isolated from Rhodiola Rosea, has been demonstrated to have multiple pharmacological activities without toxic and adverse effects. Salidroside is a promising natural remedy for further development. Currently few studies are seen to investigate the antitumor activities of Salidroside against hepatocellular carcinoma(HCC). The present study aims to investigate the anti-HCC effects of Salidroside, and to preliminarily explore the possible mechanisms.Methods:The present study examined the effects of Salidroside on the proliferation and apoptosis in human HCC HepG2 cells in vitro. Firstly, MTT assay and 3H-TdR incorporation assay were used to evaluate the effects of Salidroside on proliferation in HepG2 cells, in order to determine the effective doses of Salidroside against HCC and provide evidence for subsequent mechanistic investigations. We then used Hoechst staining and flow cytometry analyses to determine the effects of Salidroside on HepG2 cell apoptosis. We further used JC-1 staining to examine the effects of Salidroside on mitochondrial membrane potential in treated HepG2 cells. And then Western blot assays were performed to determine the expression of apoptosis-related proteins Bcl-2 and Bax, the release of cytochrome c from mitochondrion to cytoplasm, and the activities of Caspase-9 and Caspase-3 in Salidroside-treated HepG2 cells. Finally, we used Western blot assays to determine the effects of Salidroside on the protein expression of p53, and further used p53 specific inhibitor Pifithrin-α to test the association between Salidroside-induced apoptosis and p53 activation by Salidroside.Results:(1) Two kinds of proliferation assays demonstrated consistently that Salidroside could inhibit HepG2 cell proliferation in a dose-dependent manner. Salidroside at 60 μM and over this dose could lead to significant inhibitory effects on HepG2 cell proliferation.(2) Hoechst staining showed that Salidroside with the dose range of 10-40 μM could induce apoptosis in HepG2 cells. Flow cytometry analyses demonstrated that the apoptotic rates in Salidroside-treated HepG2 cells were increased significantly, and that Salidroside at 40 μM could result in the apoptotic rate being 41%.(3) JC-1 staining showed that Salidroside could reduce the mitochondrial membrane potential in treated HepG2 cells in a dose-dependent manner, upregulate the pro-apoptotic Bax expression, downregulate the anti-apoptotic Bcl-2 expression, and promote the release of cytochrome c from mitochondrion to cytoplasm. Further exanimation of Caspase cascade showed that Salidroside could remarkably increase the levels of active form of Caspase-9 and Caspase-3 in HepG2 cells.(4) Exanimation of signaling pathways shod that Salidroside could increase the protein expression of p53 in HepG2 cells. Use of p53 specific inhibitor PFT-α found that pharmacological inhibition of p53 could abrogate the Salidroside reduction in expression of anti-apoptotic Bcl-2 and also diminished the Salidroside upregulation of pro-apoptotic Bax. Besides, PFT-α also significantly abolished the Salidroside upregulation of active Caspase-3 in HepG2 cells.Conclusions:The current investigations showed that Salidroside could inhibit cell proliferation and effectively induce apoptosis in HepG2 cells in vitro. These effects were associated with upregulation of p53 and activation of mitochondrial apoptosis pathway leading to activation of Caspase cascades in Hep G2 cells. These events could contribute to the molecular mechanisms underlying the anti-tumor activities of Salidroside. These discoveries could provide experimental evidence for developing Salidroside as a promising anti-HCC agent with low toxicity.